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Home , Andrology, Semen

Journal of Life Sciences 10 (2016) 91-99 doi: 10.17265/1934-7391/2016.02.005 D DAVID PUBLISHING

Identification of 11 STD in Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology

Rubina Ghani1, Kashif Nisar1, Hasan Ali2 and Saara Ahmad3 1. Department of Biochemistry, Jinnah Medical & Dental College, Pathological & Molecular Laboratories Karachi, Pakistan 2. Department of Biochemistry, Bahria University, Bahria Medical & Dental College 3. Department of Biochemistry, Baqai Medical University, Pathological & Molecular Laboratories Karachi, Pakistan

Abstract: The of sexually transmitted (STI) pathogens from an infected donor to the recipient of a semen donation in assisted conception may result not only in acute infection but also in long-term reproductive complications or adverse outcomes of including infection of the offspring. Semen samples were obtained by into sterile containers. Samples were subjected to within one hour of collection and processed for freezing within two hours of collection. The motility was determined. After DNA extraction the PCR was performed and amplicons are subsequently hybridized to -specific capturing probes via “Flow-through” hybridization. During our study we came across with the STI pathogens present in semen and main cause of was note. It was also observed that route for the transfer for these STI pathogens were the men working in other cities and visited commercial workers and their complained for infertility. We have reported our data that after the normal sperm count in semen samples of men with infertility or subfertility they were infected with Chlamtdia trachomatis, Neisseria gonorrhoeae, and Mycoplasma hominis. In our study all the 11 pathogens were detected which cause serious reproductive complications and infection in their offspring.

Key words: Sexually transmitted disease, semen analysiss, chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Human Pappilloma virus.

1. Introduction problem with severe medical and psychological consequences for millions of men and women [7]. The A variety of sexually transmitted infectious (STIs) number of STIs is increasing throughout the world of the male genitourinary (GU) tract have been despite diagnostic and treatment advances in recent associated with male factor infertility. In both men years [8]. and women these may result in significant More than 340 million cases of curable sexually morbidity and make a formidable contribution to transmitted infections (STIs) including Treponema world-wide health expenditure [1-5]. Infertility is the pallidum, Neisseria gonorrhoeae, Chlamydia failure of a couple to become pregnant after one year trachomatis and Trichomonas vaginalis were of regular, unprotected intercourse. About a third of estimated to have occurred worldwide in 1995 [9]. infertility problems are due to infertility, and Previous studies have shown that the presence of other another third are due to . In the concomitant STIs increases the likelihood of HIV remaining cases, infertility affects both partners or the transmission [10-13]. cause is unclear [6]. Sexually transmitted infections As in other developing countries, sexually (STIs) are recognized as a major transmitted infections (STIs) and reproductive tract infections (RTIs) represent a major public health Corresponding author: Rubina Ghani, M.Sc., Ph.D., research fields: infectious disease and molecular diagnosis. problem in Bangladesh [6]. Control of ulcerative

92 Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology

(syphilis, chancroid, and virus type 2 into a semen donation program [14-18]. [HSV-2] infection) and nonulcerative (gonorrhea, The objective of screening semen samples before chlamydia, and trichomoniasis) STIs and of RTIs they are used for assisted reproduction procedures is (bacterial vaginosis and candidiasis) is important not to protect the recipients of semen donations and their only for preventing complications related to infection offspring from bacterial and viral STIs and their but also for preventing heterosexual transmission of sequelae by preventing the transmission of STI human immunodeficiency virus (HIV). pathogens from them to the recipient (partners). The STDs are not only a cause of acute morbidity in transmission of STI pathogens to the recipient of a adults but may result in complications with sequelae semen donation may result not only in acute infection, such as infertility in both men and women, ectopic but also in long-term reproductive complications in the pregnancy, cervical cancer, premature mortality, recipient and possible adverse outcomes of pregnancy, congenital syphilis and fetal wastage, low birth weight, including infection in the offspring [19-21]. and prematurity and ophthalmia neonatorum. The STDs that are caused by bacterial, mycological and 2. Material and Method protozoal agents have been curable by appropriate 2.1 Specimen Collection and chemotherapeutic agents for more than 40 years. In spite of this, such STDs have The duration of this study was for two months i.e. continued to be a public health problem in both from November2013 to January 2014 and all the semen industrialized and developing countries. In many samples were collected with history and cause of developing countries STDs have for several decades infertility. During this study, the history form was filled ranked among the top five diseases for which adults and we found that most of the were driver and seek services. Reliable surveillance is their family were settle in small village or Goth areas rarely in place and the exact magnitude of the problem people settle in Karachi not only came from four is frequently unknown. provinces but from sample villages in Sindh for jobs. The goal of estimating correctly a man’s fertility In this study samples were selected from different potential has long been of great interest to researchers. , maternity homes and medical centers from The term ‘male infertility’ does not constitute a different parts of Karachi. As Karachi is the defined clinical syndrome but rather a collection of cosmopolitan city and all the people from different different conditions exhibiting a variety of etiologies parts of country come here for the business and its and varying prognoses [6]. economy is based on industrial production, trade and The present guidelines are intended for laboratories finance. As well as it is the commercial and industrial involved in the testing of semen samples to ensure, capital of the country. within the limitations of existing laboratory methods, The semen sample was distributed into four parts that the semen samples are free from pathogens that and send to each department for the basic test. The can cause sexually transmitted infections (STIs). serology, culture and routine semen analysis test were Testing of semen specimens for STI pathogens is not performed and data was collected, whereas with one recommended as a means of diagnosis of clinical part of the sample was used for the DNA extraction. syndromes in donors of assisted conception programs, DNA extraction: The DNA purification from whole nor should these specimens be used in tests of cure collected in EDTA was carried out by using following treatment. Donors generally undergo Epicenter Kit (cat# MCD85201) and the protocol was vigorous screening for STIs before they are accepted followed accordingly.

Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and 93 “Flow-through” Hybridization Technology

2.2 PCR Amplification and Gene Flow Hybridization placed into thermal cycler and run following the thermal condition in table 2 below The PCR was performed. The Amplification Control After amplification the sample tubes were cooled (signal at B5) is used to monitor the presence of PCR and were prepared for the hybridization following inhibitors and the presence of sufficient amount of steps given in the table 3. extracted human cellular DNA content during PCR amplification. Absence of Amplification Control signal 3. Result indicates either failure of the PCR amplification The 60 semen analysis samples were collected from (presence of PCR inhibitor) or insufficient amount of a lab and the basic screening was done in which sperm DNA (or absence of specimen) during PCR morphology and its motility was checked according to amplification. This control signal should always be WHO guide line as shown in table 4. positive with a clear visible dot in every tested sample. Then the PCR was performed on those samples The PCR reaction was prepared according to whose count was normal and there was infertility. The protocol given table 1 and the final volume was made Table 1 Shows the protocol to make master mix for PCR upto 25 µL. amplification. Aliquot 20 µL of Master Mix (containing PCR Component Volume (µl) Reaction Mix, 25X Primer Mix and DNA Taq PCR Reaction Mix 18.6 Polymerase) into each PCR tube and add appropriate 25X Primer Mix 1.0 amount of DNA template suggested. Top up the DNA Taq Polymerase 0.4 reaction volume to 25 µL with DNase Free Water DNA Template Up to 5.0 µL when necessary. Mix and spin down the PCR reaction DNase Free Water Variable mixture (including the sample). PCR tubes were Total 25.0

Table 2 Shows the thermal cycle condition according to which the DNA is amplified. Stage Step Temperature (°C) Time Hold Initial denaturation 95 10 min Denaturation 95 30 sec 43 cycles Annealing 60 30 sec Extension 72 60 sec Hold Final extension 72 7 min Hold Final hold 4 ∞

Table 3 Shows the protocol for the flow through hybridization at a glance with FT Pro Flow-through. Hybridization-at-a-glance Solution Volume (µl) Incubation After incubation 43 °C FT pro Pre-hybridization 150 2 min Drain Hybridization 150 + 25 PCR product 5 min Drain Hybridization solution wash 200 x 3 - Drain 25 °C Blocking 150 5 min Drain Conjugate 150 5 min Drain 36 °C Wash A solution 200 x 4 - Drain Detection Solution 150 3 min Drain Wash A solution 200 x 3 Drain Stop 150 1 min Drain

94 Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology

Table 4 Shows the basic analysis performed on semen samples to filter the sperm morphology. Motility Normal Watery Viscous Viscosity ≤ 20% ≥ 40% ≥ 40 Mid Piece & Neck Teratozoo-spermia Sperm Morphology Normal Form Head Defective Tail Defective Defective index ≤ 20 ≥ 30 ≥ 20 ≥ 10 ≥ 2:1 Culture / Sensitivity Debris Round cell Epithelial cells Erythrocytes Moderate or Heavy ≥ 10 % ≥ 4% Present Agglutination Normal Head to Head Head to Tail Tail to Tail ≤ 10 ≥ 20 ≥ 10 ≥ 10

Major sexually transmitted disease pathogens detected in semen

Bacteria Viruses Protozoa

Fig. 1 These are the major sexually transmitted pathogens which are detected in semen. gene Flow-Through array test was performed and 11 The different percentage of STD identified in the pathogens were detected the semen those causing samples is shown in pie diagram Fig. 3 and table 6. infertility shown in Fig. 2 (the photograph of cassette). During our study we also came across with the In table 5 the pathogen probes and identification of co-infection which Chlamydia trachomatis, neisseria major STD pathogens causing infertility which were gonorrhoeae, mycoplasma genitalium, ureaplasma detected in during this study. urealyticum/ureaplasma as shown in Fig. 4. When molecular analysis was carried out table 5 4. Discussion was used for the identification of STD probes placed on the cassette. All the 11 pathogens probes can be Male factor infertility can be caused by abnormalities identified for each patient in one go. in the shape and size of the sperm. As with low sperm The table 5 shows the position of pathogen probe count, at least two-thirds of the sperm in the ejaculate placed in the membrane on which the amplified must be of an adequate shape and size in order to be product is hybridized with 35 to 40 min. considered normal. Male infertility can result when During our study we detected Chlamydia trachomatis, the shape prevents it from having normal neisseria gonorrhoeae, mycoplasma genitalium, mobility or strength to penetrate the ovum. In fact, ureaplasma urealyticum/ureaplasma parvum, some specialists believe that abnormal sperm shape has mycoplasma hominis, trichomonas vaginalis and an even greater effect on male fertility than low motility human papilliomas virus 6/11 in the semen samples or (low sperm count). Some of the which were having low count and infertility problems causes of abnormal semen, such as STDs, infection, detected by gene Flow-through as shown in Fig. 2, retrograde , and the ability of the ejaculate which were identified according to the in table 5. to clot properly, can significantly affect male fertility.

Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and 95 “Flow-through” Hybridization Technology

Fig. 2 Shows the target pathogens detected by Gene Flow-Through from semen sample.

Table 5 Shows the identification panel of STD probes placed on membrane.

Major STD detected in Semen Samples

2.08 2.08 CT

6.25 NG

MG

4.16 UU/UP

MH 2.08 TV

20.83 HP 10.41

Fig. 3 This shows the percentage of target pathogens detected by Gene Flow-Through from Semen sample.

96 Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology

Table 6 Shows the different STD detected from semen analysis by Gene Flow-Through to for the cause of infertility. NAME OF THE STD (N = 48) NO. DETECTED PERCENTAGE Chlamydia trachomatis (CT) 03 6.25 Neisseria gonorrhoeae (NG) 02 4.16 Mycoplasma genitalium (MG) 01 2.08 Ureaplasma Urealyticum (UU)/Ureaplasma parvum (UP) 05 10.41 Human Papillomavirus (HPV) 01 2.08 Mycoplasma hominis (MH) 10 20.83 Trichomonas vaginalis (TV) 01 2.08 Positive Control (PC) 01 2.08

Co-infection MH-UU/UP CT-UU/UP MG-UU/UP 2.08 CT-NG

4.16

2.08

2.08

Fig. 4 The distribution pie diagram shows the co-infection rate detected in the semen samples.

Sexual transmission via penile-vaginal, penile-rectal, among young couples [23]. We designed our study to or penile-pharyngeal contact accounts for the vast investigate the prevalence of 11 pathogens, in the majority of cases. The most important means of semen of healthy fertile and infertile males, using a non-sexual transmission is from mother to child sensitive Gene Flow Through technology. Our study during birth. The gonococcus is a survivor. Despite its showed that genital mycoplasmas and ureaplasmas being too fragile to be transmitted by food, water, air, were the most prevalent uropathogens in the semen of or (with very rare exceptions) fomites, it remained a infertile men and were found in asymptomatic fertile very common infection in most of the world in the men in almost equal proportion, although M hominis preantibiotic era despite sometimes strenuous efforts was significantly more prevalent that is 20.83 percent to control it. The introduction of antimicrobials led to in fertile than infertile men. The second most control of gonorrhea in some populations, but despite prevalent microorganism detected was ureaplasma the natural susceptibility of the gonococci to low urealyticum/ureaplasma which was 10.41 percent. concentrations of a wide variety of antimicrobials, 75 Male factor infertility in the majority of the cases years after the discovery of penicillin gonorrhea remains remains asymptomatic and with unknown causes. a common infection in much of the world [22]. In our clinical studies besides mycoplasma inci- The demand for assisted reproduction has increased dence was raised the other pathogen such as over the last few years because of infertility problems Trichomonas vaginalis, Chlamydia trachomatis or

Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and 97 “Flow-through” Hybridization Technology

Neisseria gonorrhea were also detected. Maleki et al. individuals may carry lower amount of organisms [31]. in 2013 [24] reported that strong relation between the Besides, real time PCR is easier and has higher presence of the studied M. hominis and U. urealyticum sensitivity and specificity. Thus, real time PCR may with urogenital infection in the under study in be the technique of choice for bacterial detection and comparison with those of control groups. quantification in semen specimens of asymptomatic In previous study, the role of genital tract male partners. Our study demonstrated that C. microorganisms as an important etiological factor in trachomatis seems to be the less spread sexually male infertility has been reported [25]. Whereas transmitted pathogen among male partners of infertile Friberg and Gnarpe in 1974 had earlier demonstrated couples. the preponderance of ureaplasmas in the semen of Infertility caused by Chlamydia was a big problem infertile men (76%) compared with fertile men (19%) for women, the infection can cause scarring in the [26]. Fallopian Tubes which can block them and mean that In many literature reports, U urealyticum and C the egg and sperm have a reduced chance of meeting. trachomatis are the most common pathogens The infection is less reported in men, it is a significant associated with male infertility [27-29]. However, our issue as it can reduce the production of sperm (by up to study not only demonstrated an overall high 80%) and cause sperm fragmentation which means that prevalence of U urealyticum (10.41%), and M the long tail of sperm may break and the genetic hominis (20.83%) but also showed that they were material of the sperm is not as tightly packed [32]. co-infected in widespread among asymptomatic Untreated chlamydia infections can also cause infertile and fertile males. During this study we also NSU—Non specific urethritis in men, which means detected other STD which were Chlamydia that you may experience pain in the genital region, trachomatis (6.25 percent), Neisseria gonorrhoeae discomfort when urinating and/or having sex. This can (4.16%), Human Papillomavirus (2.08%) and happen even if there are no symptoms in the early Trichomonas vaginalis (2.1 percent). stages of infection. In 2004 first national STI study in Pakistan was It has also been reported that that C. trachomatis conducted in order to determine the disease burden of infection could affect sperm mitochondrial function. HIV and STIs. The study showed a high burden of Caspase activity has been shown to be present in STIs among women selling sex, with the following human sperm [33-35]. Furthermore, in infertile men a prevalence; T. pallidum 7%, N. gonorrhoeae 12%, C. higher percentage of sperm with activated caspases trachomatis 11% and T. vaginalis 19%. The study also was found, confirming the existence of a highlighted a low level of knowledge about AIDS caspase-dependent apoptotic pathway in ejaculated among the women selling sex; 24% had never heard human sperm [36, 37]. In the third part of our study, of AIDS and 26% had not used a with their we studied the activation of caspase 3 in spermatozoa recent . In Pakistan, the terms HIV and of infertile men. We noticed also a significant increase AIDS are commonly used synonymously. of caspase 3 activation in male partners of infertile The importance of genital tract microorganisms as couples with C. trachomatis DNA in semen specimens an etiologic factor in male infertility is still a in comparison to male partners of infertile couples controversial topic [30]. The purpose of this study was without C. trachomatis DNA in semen specimens. So to determine the prevalence of several common public health are concerned about chlamydia sexually transmitted pathogens among male members infections because of the effect if can have on fertility. of infertile couples. Asymptomatically infected This can damage individual’s mental health and well

98 Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology being, and cost thousands of pounds in fertility Reference treatment. Preventing the infection at an early stage is [1] Haidl, G., Allam, J. P., and Schuppe, H. C. 2008. the key to reducing these later issues. Public health “Chronic : Impact on Semen Parameters and also wants to protect against the costs of treating NSU Therapeutic Options.” Andrologia 40 (2): 92-6. in the future. [2] Comhaire, F. H., De Kretser, D., and Farley, T. M. M. 1987. “Towards More Objectivity in the Management of During our study co-infection detected was U Male Infertility.” Int. J. Androl. 10: 1. urealyticum and M hominis was 4.8 percent, C. [3] Dohle, G., Colpi, G., Hargreave, T., Papp, G., Jungwirth, trachomatis and Neisseria gonorrhoeae was 2.1 A., and Weidner, W. 2005. “EAU Guidelines on Male percent as shown in figure 4. It was reported as Infertility.” Eur. Urol. 48 (5): 703-11. [4] Pellati, D., Mylonakis, I., Bertoloni, G., Fiore, C., co-infection which was important STD screening Andrisani, A., and Ambrosini, G., et al. 2008. “Genital parameters. It has been reported that N. gonorrhoeae Tract Infections and Infertility.” Eur. J. Obstet. Gynecol. infection is asymptomatic and often mistaken as Reprod Biol. 140 (1): 3-11. [5] Smith, W. B. I. I., Trost, L. W., Chen, Y., Rosenchans, A., virginal or bladder infection and may progress to and Hellstrom, W. T. G. 2014. “Male Infertility: A serious complications as skin pustules or petechial, Complete Guide to Lifestyle and Environmental Factors.” septic arthritis, memingitis or endocarditis [38]. Chapter 9 Springer, Science, New York 127. [6] American Urological Association. The Optimal 5. Conclusions Evaluation of the Infertile Male. AUA Best Practice Statement. Revised 2010. The results of our study demonstrate that the genital [7] Agacfidan, A., and Kohl, P. 1999. “Sexually Transmitted mycoplasmas and ureaplasmas seem to be widespread Diseases (STDs) in the World.” FEMS Immunol. Med. among male partners of infertile couples as previously Microbiol. 24 (4): 431-5. [8] Hashwani, S., Hiran, T., and Fatima, M. 1999. reported in Tunisia. The study of the comparison of “Awareness of Sexually Transmitted Diseases in a the semen parameters of infertile men with and Selected Sample in Karachi.” J. Pak. Med. Assoc. 49: without genital ureaplasmas and mycoplasmas has not 161-4. shown any significant differences, apart from the [9] WHO: Global Strategy for the prevention and control of Sexually Transmitted Infections: 2006-2015. Breaking sperm concentration in the infection of M. hominis the chain of transmission WHO; 2007. and M. genitalium and sperm morphology in the [10] White, R. G., Orroth, K. K., and Glynn, J. R., et al. 2008. infection of M. hominis. Our results also indicate that “Treating Curable Sexually Transmitted Infections to Prevent HIV in Africa: Still an Effective Control PCR-and Gene Flow through hybridization assay Strategy?” J. Acquir. Immune Defic Syndr 47 (3): 346-53. method provides a rapid and cost effective measure to [11] Sangani, P., Rutherford, G., and Wilkinson, D. 2004. detect human genital mycoplasmas, ureaplasmas, “Population-based Interventions Forreducing Sexually chlamydia trachomatis and neisseria gonorrhoeae Transmitted Infections, Including HIV Infection.” Cochrane Database Syst. Rev. 2: CD001220. which are useful for etiological and epidemiological [12] Cohen, M. S. 1998. “Sexually Transmitted Diseases studies of these pathogens. Little information was, Enhance HIV Transmission: no longer a Hypothesis.” however, available regarding the effect of these STI Lancet 351 (3): 5-7. on the sperm quality, as well as their relationship with [13] Mohsin, S. K., Magnus, U., Shakila, Z., and Cecilia, S. L. 2011. “HIV, STI Prevalence and Risk Behaviours among the leukocyte count. Therefore, it can be concluded Women Selling Sex in Lahore, Pakistan. Khan et al.” that the screening of these in routine semen BMC Infectious Diseases 11: 119. analysis is not clinically relevant in our specific [14] British Society. British Andrology society population. It should be restricted for men undergoing guidelines for the screening of semen donors for donor 1999. Liesnard CA. Screening of semen complete evaluation of infertility, genital infection and donors for infectious diseases. Hum. Reprod 14: male partners from couples undergoing IVF. 1823-6.2.

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